Salmonella typhimurium persists within macrophages in the mesenteric lymph nodes of chronically infected Nramp1+/+ mice and can be reactivated by IFNgamma neutralization

Abstract

Host-adapted strains of Salmonella are capable of establishing a persistent infection in their host often in the absence of clinical disease. The mouse model of Salmonella infection has primarily been used as a model for the acute systemic disease. Therefore, the sites of long-term S. typhimurium persistence in the mouse are not known nor are the mechanisms of persistent infection clearly understood. Here, we show that S. typhimurium can persist for as long as 1 yr in the mesenteric lymph nodes (MLNs) of 129sv Nramp1(+)(/)(+) (Slc11a1(+)(/)(+)) mice despite the presence of high levels of anti-S. typhimurium antibody. Tissues from 129sv mice colonized for 60 d contain numerous inflammatory foci and lesions with features resembling S. typhi granulomas. Tissues from mice infected for 365 d have very few organized inflammatory lesions, but the bacteria continue to persist within macrophages in the MLN and the animals generally remain disease-free. Finally, chronically infected mice treated with an interferon-gamma neutralizing antibody exhibited symptoms of acute systemic infection, with evidence of high levels of bacterial replication in most tissues and high levels of fecal shedding. Thus, interferon-gamma, which may affect the level of macrophage activation, plays an essential role in the control of the persistent S. typhimurium infection in mice.

Figure 1.

Systemic infections in 129sv mice induce splenomegaly and enlarged MLN. (A) The spleen from an uninfected age-matched 129sv mouse on the left is shown next to the spleen from a 129sv mouse (spleen on the right) infected with SL1344 for 60 d. (B) MLN from the same mice as in A (right, infected MLN). (C) The weights of the spleens are plotted on the y axis, with time from infection on the x axis. Infected spleens weighed significantly more than the uninfected control spleens. P < 0.0001 for day 30; P = 0.01 for day 140; P = 0.01 for day 365 (n = 5 for each day).

Figure 2.

Shedding of S. typhimurium in feces from persistently infected 129sv mice. A mean of 0.1 g of feces was collected from five different mice on days 114–117 after infection with SL1344. The contents were homogenized and plated on XLD plates. The CFU per gram of feces varied from day-to-day in a given mouse.

Figure 3.

Mean anti–S. typhimurium IgG titers in persistently infected 129sv mice. S. typhimurium-infected (n = 5 for days 60 and 140; n = 4 for days 180 and 270; n = 13 for day 365) and uninfected age-matched control mice (n = 3, except for day 180). Antibody titers were determined by ELISA using killed whole S. typhimurium sonicate as antigen. (Closed diamonds) IgG titer in S. typhimurium-infected mice. (Open triangles) IgG titer in uninfected controls. Each serum was tested at least three times in triplicate.

Figure 4.

Histology of infected tissues from mice persistently infected with S. typhimurium. Liver and spleen sections were stained with hematoxylin and eosin. (A) Liver section from mouse infected for 60 d. Arrows point to multiple areas of inflammation and microgranulomas. (B) Liver section form mouse infected for 365 d. Arrow points to a focal granuloma. (C) Section of spleen infected for 60 d. Arrow shows accumulation of PMN in red pulp. (D) Section of spleen infected for 140 d. Arrow shows accumulation of macrophages. (E) Section of liver infected for 365 d. Bars: (A and B) 0.1 mm; (C–E) 20 μm.

Figure 5.

Monitoring 129sv mice persistently infected with S. typhimurium by an IVIS. 129sv mice were inoculated by oral administration of the bioluminescent labeled wild-type S. typhimurium strain, SL1344hha::Tn5lux at a dose of 10⁸ CFU and monitored over 80 d. Days are indicated in the top left corner of each image. Light intensity is represented by a color scale in counts. Eight mice were inoculated and imaged on days 7, 23, 40, and 80. The arrow points to a mouse 2, which showed variable levels of signal over the 80 d. Mouse 4 died between 40 and 80 d. The asterisk indicates mice that were killed for analysis of MLN by confocal microscopy (e.g., mouse 2, 5, and 7).

Figure 6.

S. typhimurium persist inside of macrophages within the MLN of chronically infected mice. (A) MLN sections were labeled with anti-Salmonella antibody (green), antineutrophil antibody, Gr-1 (red), and an anti–mouse IgG antibody (blue) that nonspecifically stains other immune cells. (A, D, and E) The image is a projection of a 20-μm z-stack collected through the 60× objective on a confocal microscope (model MRC-600; Bio-Rad Laboratories). Arrow points to bacterium. Bar, 10 μm. (B) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody, MOMA-2 (red), and ToTo-3 to stain all nuclei (blue). The image is a projection of a 15-μm z-stack collected through the 40× objective on a confocal microscope. (C) Volocity 2.0 was used to project the side view (yz) and bottom view (xz) of the infected macrophage in B (represented by the xy view), which together demonstrate the intracellular location of the bacterium. Bar, 5 μm. (D) Three-dimensional projection of macrophage in C. (E) MLN sections were labeled with anti-Salmonella antibody (green), antimacrophage antibody (red), and B220 (blue), the anti–B lymphocyte antibody. Bar, 10 μm.

Figure 7.

Anti-IFNγ neutralizing antibody reactivates S. typhimurium replication in persistently infected mice. (A) Bacterial counts in tissues from 129sv mice infected for 260 d and injected with IFNγ neutralizing antibody. (B) Bacterial counts in tissues from 129sv mice infected for 260 d and injected with isotype control antibody. The x axis is the tissue where F = feces, C = cecum, PP = Peyer's patches, M = mesenteric LNs, S = spleen, L = liver, G = gall bladder. The y axis denotes individual mice. The z axis is the CFU per gram of tissue. The Mann-Whitney U test was performed for statistical significance comparing the bacterial load from anti-IFNγ neutralizing antibody–treated mice with control antibody-treated mice. *, P ≤ 0.05; **, P ≤ 0.01.

Figure 8.

Histology of infected tissues from mice persistently infected with S. typhimurium and treated with IFNγ neutralizing antibody. (A) Liver, (B) lung, and (C) heart. Sections were stained with hematoxylin and eosin from a mouse infected for 260 d followed by 3 wk of IFNγ neutralizing antibody. (A) Arrow points to area of inflammation and microgranulomas. (B) Arrow points to inflammation in the pleura. (C) Arrow points to inflammation covering the epicardium. (D) Liver and (E) heart. Bacteria in sites of inflammation from serial sections were stained with antibody to S. typhimurium (green), actin was stained with phalloidin-Alexa594 (red), and nuclei of host cells were stained with Toto-3 (blue). (D) Arrow points to bacterium in the center of lesion. (Inset) Enlargement of the area of lesion containing the bacterium. (E) Arrow points to bacterium in the epicardium. (Inset) Enlargement of the area. Bars: (A) 20 μm; (B and C) 200 μm; (D and E) 100 μm.